Cy0 is still the first method in Real-time PCR analysis that does not require the assumption of equal efficiency between unknowns and standard curve.As shown, the Cy0 method and the Gold Standard Ct (and others) were equally precise and accurate in optimal amplification conditions, while, when PCR efficiency was decreases, diluting amplification mix quantity or adding a biological inhibitor such as IgG, the Cy0 method gave significantly more accurate and precise quantification.
To have a look to a wider and deeper analysis regarding the reliability of Cy0 see
Evaluation of qPCR curve analysis methods for reliable biomarker discovery: Bias, resolution, precision, and implications (FREE for Cy0method.org subscribers only under kind permission of Elsevier Limited - license #3036951067977) (p.s. Thanks to dr Jan M. Ruijter for involving our project in his evaluation of qPCR curve analysis methods).
Moreover, the analysis of the data collected through this site, has given us the cue to find a path leading to a quantification method able to provide a valid result almost in every (suboptimal) amplification condition.
In this sense, every global model we dealed with, has shown fitting problems in particular shaped amplification curves, i.e. when the flex point is too close to the X-axis, or as well, dealing with Taqman chemistry curves, either Sybr Green.
Sincerely, The Cy0 Team